Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-04-08
2004-05-04
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S069100, C435S091410, C435S320100, C536S023400
Reexamination Certificate
active
06730483
ABSTRACT:
The present invention concerns phagemids for the selection of specific antibodies from large recombinant libraries, the production of these phagemids and their use to select specific antibodies from large recombinant libraries using small amounts of antigen.
Plasmid and phage antibody libraries have been established in
E. coli
from PCR amplified immunoglobulin families following immunization. Recombinant antibodies to immunogens were selected by an ELISA assay of the bacterial supernatant from isolated bacterial colonies (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T. & Winter, G.: Binding activities of a repertoire of single immunoglobulin variable domains secreted from
Escherichia coli
. Nature 341 (1989) 544-546) or by screening nitrocellulose plaque lift-offs of bacterial colonies for reactivity to the radioactively labeled immunogen (Huse, W. D., Sastry, L., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R., Benkovic, S. J. and Lerner, R. A.: Generation of a large combinatorial library of the immunoglobin repertoire in phage lambda. Science 246 (1989) 1275-1281). However, for the selection of specific antibodies from randomly combined light and heavy chain libraries of non-immunized animals that do not contain a preponderance of antibodies to a particular antigen, a procedure is required for screening millions of antibody producing bacteria.
A possible way to screen a broad range of antibodies is to attach recombinant antibodies to the surface of bacteria or bacteriophages so that they can then be rapidly selected by antigens bound to a solid phase. Given the difficulties of targeting proteins to the cell surface of bacteria, an attractive candidate in view of its small size and relatively simple genetic make-up is the M13 family of filamentous bacteriophages (for reviews see Webster, R. E. and Lopez, J. in “Virus Structure and Assembly” ed. S. Casjens, publ. Jones and Bartlett Inc., Boston/Portala Valley, USA, 1985; Day, L. A., Marzec, C. J., Reisberg, S. A. and Casadevall, A.: DNA packaging in filamentous bacteriophages. Ann. Rev. Biophys. Biophys. Chem. 17 (1988) 509-539).
The product of gene III (pIII) is a relatively flexible and accessible molecule composed of two functional domains; an amino-terminal domain that binds to the F pilus of male bacteria during infection and a carboxy-terminal domain buried within the virion that is important for morphogenesis. Peptides can be inserted between the two domains of pIII (Smith, G. P.: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228 (1985) 1315-1317) or near the N-terminus (Parmley, S. F. and Smith, G. P.: Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene, 73 (1988) 305-318) without destroying its functions in morphogenesis and infection. After much pioneering work on the use of pIII in fd phages for carrying foreign peptides, Parmely and Smith (1988, a.a.O.) showed that peptide epitopes inserted at the aminoterminal end could bind phages to immobilized antibodies. As a consequence of this work it has been possible to generate peptide libraries that can be screened for binding to ligands and antibodies (Scott, J. K. and Smith, G. P.: Searching for peptide ligands with an epitope library. Science 249 (1990) 386-390; Devlin, J. J., Panganiban, L. C. and Devlin, P. E.: Random peptide libraries; A source of specific protein binding molecules. Science 249 (1990) 404-406; Cwirla, S. E., Peters. E. A., Barrett, R. W. and Dower, W. J. Peptides on phage, a vast library of peptides for identifying ligands.: Proc. Natl. Acad. Sci. USA, 87 (1990) 6378-6382).
McCafferty, J., Griffiths, A. D., Winter, G. and Chiswell, D. J.: Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348 (1990) 552-554 reported the assembly of an antibody-pIII fusion protein into an fd phage with a Tet
R
gene after inserting antibody DNA into the 5′ end of gene III. The phage remained infectious and was able to be enriched by affinity chromatography. However, fusion phage have been shown to be mainly useful for displaying relatively small inserts, probably, because the large inserts have an adverse effect on the infectivity function of pIII (Parmlee and Smith, 1988, a.a.O). There is a large risk, therefore, that phage libraries will quickly become dominated by deletion mutants after library amplification.
Thus the technical problem underlying the present invention is to provide a more efficient mean for screening antibody libraries in bacteria.
This problem is solved by providing a phagemid according to claim
1
that expresses a functional antibody-pIII fusion protein. Preferably the antibody is a single-chain antibody. DNA coding for an antibody-pIII fusion protein, preferably a single-chain antibody-pIII fusion protein, was incorporated into a phagemid. A major advantage of the phagemid system of this invention over McCafferty et al. (see above) is that it can be propagated as a plasmid and is not under any selection pressure to remove antibody DNA, since the expression of the fusion protein is tightly repressed. This is particularly important during the amplification of antibody libraries when faster proliferating deletion mutants could quickly dominate. The phagemid DNA, being less than half the size of the above phage DNA, also transform bacteria more efficiently. Moreover, in contrast to the above mentioned phage system, large quantities of the smaller phagemid DNA are produced and large amounts of antibody protein are available after induction, thereby greatly facilitating its analysis. Expression of the antibody-pIII fusion protein, preferably the single-chain antibody-pIII fusion protein, using the pSEX phagemid (see below) and its packaging into viral particles greatly facilitate the establishment of bacterial systems for the isolation of high affinity antibodies. Millions of antibody-producing clones from antibody libraries can now be rapidly screened by binding to immobilized antigen. A further advantage over conventional screening methods is that only small amounts of antigen are required, an important factor when the supply of a rare protein is limited. This system also offers the possibility of screening randomly mutated antibodies in order to increase their binding affinities. The procedure could be repeated many times until the desired specificity is achieved. It is now feasible for the first time to carry out large scale differential screening analyses of related cells and organisms. A substractive selection, e.g., using normal and neoplastic cells could be used to identify tumor associated antigens. The phagemid system also proves to be extremely useful for investigating the component of molecular interactions e.g. by selecting antibodies that inhibit ligand receptor binding.
Furthermore, the system of this invention is mostly useful for presenting other proteins or peptides at the surfaces of phagemid viral particles. For this purpose the DNA encoding the antibody has to be replaced with DNA of the desired polypeptide.
REFERENCES:
patent: 5849500 (1998-12-01), Breitling et al.
patent: 5969108 (1999-10-01), McCafferty et al.
patent: 5985588 (1999-11-01), Breitling et al.
patent: 6127132 (2000-10-01), Breitling et al.
patent: 6172197 (2001-01-01), McCafferty et al.
patent: 6387627 (2002-05-01), Breitling et al.
patent: WO 9209690 (1992-06-01), None
Kang et al., Proc. Natl. Acad. Sci. USA, vol. 88 (1991) pp. 4363-1366.
McCafferty et al., Nature 348, pp. 552-554 (1990).
Ward et al., Nature 341, pp. 544-546 (1989).
Cwirla et al., Proc. Natl. Acad. Sci. USA 87, pp. 6378-6382 (1990).
Scott et al., Science 249, pp. 386-390 (1990).
Breitling et al., Gene 104, pp. 147-153 (1991).
Huse et al., Science 246, pp. 1275-1281 (1989).
Day et al., Ann. Rev. Biophys. Chem. 17, pp. 509-539 (1988).
Smith et al., Science 228, pp. 1315-1317 (1985).
Parmley et al., Gene 73, pp. 305-318 (1988).
Devlin et al., Science 249, pp. 404-406 (1990).
Amit et al., Science 233, pp. 747-753 (1986).
Verhoeyen
Braunagel Michael
Breitling Frank
Dübel Stefan
Klewinghaus Iris
Little Melvyn
Deutsches Krebsforschungszentrum Stiftung des offentlichen Recht
McKelvey Terry
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